Experimental autoimmune encephalomyelitis (EAE) is a CD4+ T cell-mediated inflammatory CNS demyelinating disease that serves as a model for human multiple sclerosis (MS). Relapsing-remitting EAE (R-EAE) is induced in SJL/J mice following active immunization with encephalitogenic myelin peptides (e.g., PLP139-151, PLP178-191 or MBP84-104), or by adoptive transfer of peptide-specific T cells. Based on the relapsing-remitting course of the disease, along with our findings that relapses are due primarily to the cte novo activation of T cell responses against non-crossreactive endogenous myelin epitopes on the same or different myelin proteins (intramolecular or intermolecular epitope spreading), effective specific therapy of established disease requires tolerogenic targeting of T cells specific for spread myelin epitopes. We will test the hypothesis that enhanced efficiency of the i.v. injection of protein/peptide-pulsed, ethylene carbodiimide (ECDI)-fixed antigen presenting cells (Ag-SP) tolerogenic protocol is due to the induction of multiple direct and indirect (cross-tolerance) regulatory mechanisms, including clonal anergy/deletion of encephalitogenic Th1 cells, immune deviation, and induction of antigen-specific CD4+CD25+Foxp3+ Tregs. Specific Aim 1 of this proposal will build on our productive studies from the previous funding period, and determine the in vivo mechanisms responsible for downregulation of disease induction and progression following extrinsic induction of peripheral Ag-SP tolerance. In comparison to the i.v. or oral administration of soluble peptide, we have shown that tolerance induced by Ag-SP is highly effective and safe for both prevention of EAE and treatment of pre-established R-EAE, and a powerful tool for identification of the pathologic epitopes targeted at various stages of the disease process. The effects of tolerance at varying times during the relapsing-remitting disease process on the viability, activation state, functional status, and homing characteristics of naive and activated PLP139-151 -specific Tg CD4+ T cells will be determined. To determine the cellular mechanism(s) by which Ag-Sp-induced tolerance occurs, Specific Aim 2 will use a newly developed in vitro Ag-SP tolerance system employing naive peptide-specific Tg T cells. Direct effects of tolerogenic (Ag-SP) vs. immunogenic (pulsed viable APCs) encounter on viability, Th1/Th2/Th17 cytokine production and phenotype (i.e., ability to activate functional CD4+CD25+Foxp3+ Treg cells) will be determined. Lastly, to determine the molecular mechanism of tolerance induction, based on findings in other systems that tolerance induction alters T cell signaling, Specific Aim 3 will compare signaling events in APCs and naive T cells following tolerogenic (Ag-SP) vs. immunogenic peptide encounter. Collectively, these studies should enhance the understanding of the cellular and molecular mechanisms underlying a highly efficient extrinsic peripheral tolerance therapy effective for treatment of ongoing R-EAE and slated to be tested in a Phase l/ll clinical trial in MS patients.